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P Haffter

Publications and source records attributed to P Haffter.

62 records · Page 4Linked to original sources
PubMed1991

Functional interactions among two yeast mitochondrial ribosomal proteins and an mRNA-specific translational activator.

Expression of the Saccharomyces cerevisiae mitochondrial gene coding cytochrome c oxidase subunit III is specifically activated at the level of translation by at least three nuclear genes, PET122, PET494 and PET54. We have shown previously that carboxy-terminal deletions of PET122 are allele-specifically suppressed by mutations in an unlinked nuclear gene, termed PET123, that encodes a small subunit ribosomal protein. Here we describe additional pet122 suppressors generated by mutations in a second gene which we show to be the previously identified nuclear gene MRP1. Like PET123, MRP1 encodes a component of the small subunit of mitochondrial ribosomes. Our mrp1 mutations are allele-specific suppressors of carboxyl-terminal truncations of the PET122 protein and do not bypass the requirement for residual function of PET122. None of our mrp1 mutations has an intrinsic phenotype in an otherwise wild-type background. However, some of the mrp1 mutations cause a non-conditional respiratory-defective phenotype in combination with certain pet123 alleles. This synthetic defective phenotype suggests that the ribosomal proteins PET123 and MRP1 interact functionally with each other. The fact that they can both mutate to suppress certain alleles of the mRNA-specific translational activator PET122 strongly suggests that the PET122 protein promotes translation of the coxIII mRNA via an interaction with the small subunit of mitochondrial ribosomes.

DNA, Mitochondrial↗
PubMed1990

A genetic link between an mRNA-specific translational activator and the translation system in yeast mitochondria.

Translation of the Saccharomyces cerevisiae mitochondrial mRNA encoding cytochrome c oxidase subunit III (coxIII) specifically requires the products of at least three nuclear genes, PET122, PET494 and PET54. pet122 mutations that remove 24-67 amino acid residues from the carboxyterminus of the gene product were found to be suppressed by unlinked nuclear mutations. These unlinked suppressors fail to suppress both a pet122 missense mutation and a complete pet122 deletion. One of the suppressor mutations causes a heat-sensitive nonrespiratory growth phenotype in an otherwise wild-type strain and reduces translation of all mitochondrial gene products in cells grown at high temperature. This suppressor maps to a newly identified gene on chromosome XV termed PET123. The sequence of a DNA fragment carrying PET123 contains one major open reading frame encoding a basic protein of 318 amino acids. Inactivation of the chromosomal copy of PET123 by interruption of this open reading frame causes cells to become rho- (sustain large deletions in their mtDNA). This phenotype is characteristic for null alleles of genes whose products are essential for general mitochondrial protein synthesis. Thus our data strongly suggest that the PET123 protein is a component of the mitochondrial translation apparatus that interacts directly with the coxIII-mRNA-specific translational activator PET122.

Alleles↗
PubMed1990

A novel small-subunit ribosomal protein of yeast mitochondria that interacts functionally with an mRNA-specific translational activator.

Mitochondrial translation of the mRNA encoding cytochrome c oxidase subunit III (coxIII) specifically requires the action of three position activator proteins encoded in the nucleus of Saccharomyces cerevisiae. Some mutations affecting one of these activators, PET122, can be suppressed by mutations in an unlinked nuclear gene termed PET123. PET123 function was previously demonstrated to be required for translation of all mitochondrial gene products. We have now generated an antibody against the PET123 protein and have used it to demonstrate that PET123 is a mitochondrial ribosomal protein of the small subunit. PET123 appears to be present at levels comparable to those of other mitochondrial ribosomal proteins, and its accumulation is dependent on the presence of the 15S rRNA gene in mitochondria. Taken together with the previous genetic data, these results strongly support a model in which the mRNA-specific translational activator PET122 works by directly interacting with the small ribosomal subunit to promote translation initiation on the coxIII mRNA.

Electron Transport Complex IV↗
PubMed1989

Bent DNA is needed for recombinational enhancer activity in the site-specific recombination system Cin of bacteriophage P1. The role of FIS protein.

A series of recombinational enhancer mutants was constructed by manipulating the ClaI site between the two FIS binding sites of the Hin enhancer. These mutants include insertions from two to 12 base-pairs and two deletions of one or two base-pairs. Recombinational enhancer activity was found only with four mutants carrying either a four base-pair substitution, ten base-pair insertions or a one base-pair deletion, respectively; two other ten base-pair insertion mutants, however, were inactive, although FIS protein binding was unaffected. So, besides binding of FIS protein to its specific sites within the enhancer sequence and the correct helical positioning of these sites on the DNA, another criterion for enhancer activity must be fulfilled. DNA bending assays identify this requirement as a change of the enhancer DNA conformation, which FIS protein is able to induce and to stabilize. This conformational change of the DNA can be blocked by mutations in the central segment between the two FIS binding sites of the Hin enhancer. This sequence has special functions for the recombinational enhancer activity.

Bacteriophages↗
PubMed1989

A mutational analysis of the bacteriophage P1 cin recombinase gene: intragenic complementation.

Bacteriophage P1 encodes a site-specific recombinase, Cin, which regulates the alternate expression of tail fibre genes by inverting a DNA segment. To define regions of Cin important for the recombination process, we have isolated and characterised 24 different mutations of the cin gene. Most of these mutations affected amino acids that are highly conserved in other related recombinases. Some of these mutants complement each other in vivo. This intragenic complementation could be due to the assembly of heteromers containing both mutant proteins, suggesting that the active enzyme is at least a dimer.

Amino Acid Sequence↗
PubMed1988

Enhancer-independent mutants of the Cin recombinase have a relaxed topological specificity.

Cin is a member of the hin family of complementing site-specific recombinases which regulate the alternate expression of genes by inverting DNA segments. Common characteristics of this family of recombination systems are the requirement for an enhancer-like element in cis and the specificity for inversely oriented recombination sites on the same DNA molecule. We have isolated two mutants of the Cin recombinase which will efficiently recombine a substrate lacking the enhancer. In addition, these mutant proteins also catalyse efficient recombination between sites in direct orientation or on different DNA molecules. Both mutations are due to single amino acid substitutions at different positions in the protein and the two mutants have slightly different phenotypes. The finding that the loss of enhancer dependence is coupled to a change in topological specificity leads us to conclude that the enhancer determines the specificity of the system for DNA inversion.

Bacteriophages↗
PubMed1987

Purification and DNA-binding properties of FIS and Cin, two proteins required for the bacteriophage P1 site-specific recombination system, cin.

An Escherichia coli chromosomally coded factor termed FIS (Factor for Inversion Stimulation) stimulates the Cin protein-mediated, site-specific DNA inversion system of bacteriophage P1 more than 500-fold. We have purified FIS and the recombinase Cin, and studied the inversion reaction in vitro. DNA footprinting studies with DNase I showed that Cin specifically binds to the recombination site, called cix. FIS does not bind to cix sites but does bind to a recombinational enhancer sequence that is required in cis for efficient recombination. FIS also binds specifically to sequences outside the enhancer, as well as to sequences unrelated to Cin inversion. On the basis of these data, we discuss the possibility of additional functions for FIS in E. coli.

Base Sequence↗